Multi-substrate biochip unit

ABSTRACT

An analytical device has a housing that encloses a multi-substrate chip having a reference marker and a plurality of substrates, wherein the housing is configured such that the reference marker and the substrates are illuminated by respective light sources at different angles. Further contemplated analytical devices include a housing with a cavity in which a multi-substrate chip having a plurality of substrates is at least partially disposed, wherein at least one of the plurality of substrates is coupled to a carrier via a crosslinker that is disposed in a matrix.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 09/735,402, filed Dec. 12, 2000 now abandoned, andalso claims priority to PCT Application entitled Improved Biochip filedon Dec. 11, 2001 (inventors are Vijay K. Mahant and Fareed Kureshy)which are incorporated by reference herein.

FIELD OF THE INVENTION

The field of the invention is analytic devices and methods.

BACKGROUND OF THE INVENTION

Genomics and proteomics research made a vast number of nucleotide andpeptide sequences available for analysis. Consequently, high-throughputscreening of samples for the presence and/or quantity of a vast numberof known genes or polypeptides has gained considerable interest inrecent years. There are various devices and methods known in the art,and many of those devices and methods are adapted for screening ofmultiple nucleic acid sequences.

For example, in a relatively simple approach, Johann et al describe inU.S. Pat. No. 6,277,628 a test system in which a plurality of carrierstructures is enclosed in a capillary, and wherein at least some of thecarrier structures (e.g., glass beads) are covalently coated with abiomolecular probe. Johann's system advantageously reduces the ratio ofsample volume to test surface, thereby reducing potential delays due tokinetic effects. However, various problems arise with the use of suchsystems. Among other disadvantages, optical detection (e.g.,fluorescence) of a signal from a hybridized probe is at least to somedegree impaired by inadvertent absorption of light (e.g., excitation andemission) by the capillary. Furthermore, intrinsic optical effects(e.g., auto-fluorescence) of the capillary will likely further reducesensitivity of the assay or method. Still further, inadvertentfocusing/diffusion of incident and/or emitted light is almostunavoidable due to the strong curvature of the capillary. Moreover,assembly of Johann's test systems is relatively tedious and timeconsuming.

In another example, hybridization of target molecules from a sample toan immobilized capture probe is accelerated by electrophoreticassistance using a microchip-type device as described in U.S. Pat. Nos.5,632,957, 5,605,662, and 5,849,486. Use of such microchip devices notonly increases the speed of molecular association between a targetmolecule and a capture probe, but also allows addressability of each“pixel” of the test array. Furthermore, stringency may be electronicallyregulated in a relatively simple manner in a reverse process toelectrophoretically assisted hybridization. However, the sample densityof such devices in many commercially available systems is typicallylimited to about 100 pixels per device. Moreover, electrophoreticallyassisted hybridization requires use of complex and relatively expensivechips, and loading/hybridization and detection are typically performedusing separate instruments, thereby further increasing initial,operating, and maintenance expenses.

In a further example, test arrays are produced using a photolithographicprocess, thereby allowing relatively high density of capture probes(e.g., greater than 10000 probes per array). Systems for suchhigh-density arrays are described, for example, in U.S. Pat. Nos.5,599,695, 5,843,655, and 5,631,734. While high-density arrays areparticularly useful for sequencing or complex genetic analysis, numerousdisadvantages remain. For example, custom synthesis of such high-densityarrays is likely cost-prohibitive for all but a few individuals and/ororganizations. Furthermore, high-density arrays will often have limitedapplications in routine clinical diagnostics. Moreover, due to theparticular chemistry employed in building such arrays, non-nucleic acidprobes (e.g., receptors, antibodies, and other polypeptides) aredifficult, if at all, to implement.

Thus, although numerous multi-substrate arrays are known in the art, allor almost all of them suffer from one or more disadvantage (e.g., highcost, difficult to customize, specialized chemistry, etc.). Therefore,there is still a need to provide improved multi-substrate array devicesand methods.

SUMMARY OF THE INVENTION

The present invention is directed to an analytical device that includesa housing having a cavity, wherein a multi-substrate chip at leastpartially disposed in the cavity, and wherein contemplatedmulti-substrate chip have reference marker and a plurality of substratesin predetermined positions with at least one of the plurality ofsubstrates being coupled to a carrier via a crosslinker that is disposedin a matrix. Particularly preferred devices include a housing that isconfigured such that the reference marker and the substrates areilluminated by respective light sources at different angles.

In one aspect of the inventive subject matter, contemplated cavitiesfurther include a liquid manipulation port, wherein the cavity haspreferably a volume of between 0.01 ml and 1 ml. Still further preferreddevices include an overflow compartment, which may further include anoverflow liquid manipulation port, wherein the cavity is in fluidcommunication with the overflow compartment when the cavity contains aliquid in a volume that is greater than a predetermined volume of thecavity.

In another aspect of the inventive subject matter, contemplated devicesmay comprise a second multi-substrate chip at least partiallydisposed-in the cavity, or a second cavity and a second multi-substratechip that is at least partially disposed in the cavity.

In alternative aspects of the inventive subject matter, the cavity isformed by the housing and a base element, wherein the multi-substratechip is coupled to the base element (which is preferably configured totransfer thermal energy and/or ultrasound energy to at least one of themulti-substrate chip and a fluid). In further contemplated aspects, oneor more of the substrates are contacted with a sample fluid, a reagentfluid, a wash fluid, and/or a detection fluid when the multi-substratechip is in a substantially horizontal position, it is further preferredthat binding of an analyte to a substrate is also detected while themulti-substrate chip is in a substantially horizontal position.

In still further contemplated aspects, the multi-substrate chip and/orthe housing include a reference marker that is automatically readable,and contemplated matrices will further include at least one additiveselected from the group consisting of a buffer, a humectant, a lightblocking agent, and a surfactant. Suitable matrices may include a singlelayer, or the matrix maybe formed from at least two chemically distinctlayers. A plurality of contemplated analytical devices may be stored ina magazine, preferably such that the base element of the first device isabove the housing of the second device.

Various objects, features, aspects, and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention, along with theaccompanying drawing.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A is a perspective view of an exemplary multi-substrate testdevice.

FIG. 1B is a schematic vertical cross-sectional view of a portion of themulti-substrate test device of FIG. 1A.

FIG. 2A is a schematic top view of one alternative multi-substrate testdevice.

FIG. 2B is a schematic top view of another alternative multi-substratetest device.

FIG. 3 is a schematic side view of a magazine including a plurality ofmulti-substrate test devices.

FIG. 4 schematically illustrates illumination of an exemplarymulti-substrate test device having an overflow compartment.

DETAILED DESCRIPTION

The inventors have discovered that a multi-substrate test device may befabricated in a conceptually simple and cost effective manner. Moreover,particularly, contemplated multi-substrate test devices allow simplecustomization of the array, fast read-out times, significantly reducedphotobleaching of fluorophores, and the substrates are not limited to aparticular group or class of biomolecules.

As used herein, the term “multi-substrate” refers to a plurality ofchemically and/or physically distinct molecules, wherein the number ofsuch molecules is generally between two and several ten thousandmolecules, more typically between hundred and several thousand, and mosttypically between one hundred and one thousand. Contemplated substratesinclude biological (i.e., naturally occurring) and non-biological (i.e.,synthetic) molecules, wherein especially contemplated biologicalmolecules include nucleic acids (e.g., DNA, mRNA, hnRNA, snRNA, etc.),polypeptides (e.g., enzymes, receptors, antibodies, cytokines,structural proteins, etc.), lipids (membrane lipids, messenger lipids,lipoprotein-bound lipids, etc.), carbohydrates (e.g., glycocalixcarbohydrates, glycogen, etc.), and all combinations and/or fragmentsthereof

As further used herein, the terms “first angle” and “second angle” referto an angle formed between the surface of the (matrix of the)multi-substrate chip and the incident light beam where the incidentlight beam is either focused or a laser beam. Where the incident lightis diffuse or diffracted, the angle is formed between a straight linebetween the surface of the (matrix of the) multi-substrate chip and theportion of the light emitting device closest to the surface of the(matrix of the) multi-substrate chip, wherein the light emitting deviceis a light bulb, light-emitting diode, arc, or electroluminescentsource.

Examples for non-biological molecules include synthetic nucleic acids,which may further include modified nucleosides or nucleotides (e.g.,DNA, MRNA, hnRNA, snRNA, etc.), natural and synthetic polypeptides,which may further include modified amino acids (e.g., enzymes,receptors, antibodies, cytokines, structural proteins, etc.), syntheticlipids (membrane lipids, messenger lipids, lipoprotein-bound lipids,etc.), synthetic carbohydrates (e.g., glycocalix carbohydrates,glycogen, etc.), and all combinations and/or fragments thereof.

The term “chip” as used herein refers to a carrier that has a pluralityof substrates in predetermined positions, wherein at least one of thesubstrates is coupled to the carrier via a crosslinker that is disposedin a matrix. Particularly contemplated multi-substrate chips aredescribed in commonly-owned and copending U.S. patent application Ser.No. 09/735,402, filed Dec. 12, 2000, and priority PCT Applicationentitled Improved Biochip filed on Dec. 11, 2001 (inventors are Vijay K.Mahant and Fareed Kureshy) which is incorporated by reference herein.

As still further used herein, the term “predetermined position” of asubstrate refers to a particular position of the substrate on the chipthat is addressable by at least two coordinates relative to a referencepoint on the chip, and particularly excludes a substantially completecoating of the chip with the substrate. Therefore, preferred pluralitiesof predetermined positions will include an array with a multiple rows ofsubstrates forming multiple columns (e.g., each substrate has ax-coordinate and a y-coordinate, with x and y greater than 1).

An exemplary multi-substrate test device 100 is depicted in FIG. 1A. Thetest device 100 has a housing 110, in which a cavity 120 is formed.Cavity 120 typically has a volume of between about 0.01 ml and 10 ml. Aliquid manipulation port 122 is in fluid communication with the cavity120, and the cavity is further partially surrounded by overflowcompartment 124 that receives liquid from the cavity when the cavitycontains liquid in a volume that is greater than the volume of thecavity. The overflow liquid manipulation port 125 is disposed on theopposite side of the liquid manipulation port 122 and in fluidcommunication with the overflow compartment 124. A multi-substrate chip130 is coupled to base element 140, which forms together with thehousing 110 the cavity 120. Base element 140 further includes a guideelement 142 on at least two sides.

FIG. 1B depicts a schematic vertical cross-sectional view of a portionof the multi-substrate test chip in which a matrix 138 (comprising afirst layer 138A and a second layer 138) is coated onto a carrier 134.Embedded within the first layer 138A of matrix 138 is a plurality ofcrosslinkers 136 to which a plurality of substrates 132 are coupled(here: via molecular tethers (lines between crosslinkers andsubstrates)). Also disposed in a predetermined position on the matrix isan reference marker 150 and 150′, which are also coupled via acrosslinker (and molecular tether) to the first layer of the matrix.

With respect to the housing 110, it is generally preferred that thehousing is manufactured from a transparent high-density polyethylene.However, it should be appreciated that the material for the housing mayvary considerably, and alternative materials include natural andsynthetic polymers, metals, ceramics, glass, pressed paper, and anyreasonable combination thereof. For example, where it is preferred thatthe housing is disposable, various synthetic polymers and/or pressedpaper are considered particularly suitable. On the other hand, andespecially where the housing will be reused several times, more durablematerials (e.g., ceramics or metal) may be advantageously employed.Furthermore, contemplated materials may also included to allow certainprocessing steps that would otherwise damage the housing. For example,where the housing needs to be sterilized (e.g., by radiation orautoclaving), glass may advantageously be employed as a housingmaterial.

Similarly, optical characteristics need not necessarily be limited to atransparent material. For example, where desired a reflective or lightabsorbing material may be employed to improve assay sensitivity.Furthermore, the housing may (by itself or in combination with the baseelement) be employed to transfer energy to and from the sample. Forexample, the base portion of the housing may be employed as a transducerfor ultrasound energy, while the remainder of the housing may be adaptedto heat and/or cool the sample disposed in the housing.

However, it is especially preferred that at least a portion of thehousing comprises a clear, semi-transparent, or translucent (i.e.,light-permeable) portion that allows incident light to pass through thehousing. Such housings are particularly desirable where themulti-substrate chip includes a reference marker and at least one lightsubstrate. Thus, especially preferred devices comprise a housing atleast partially enclosing a multi-substrate chip that includes areference marker and a plurality of substrates in predeterminedpositions, wherein the reference marker is illuminated by a first lightsource (180) at a first angle (182), and wherein at least one of theplurality of substrates is illuminated by a second light source (170) ata second angle (172), and wherein the housing is configured such thatthe first angle and the second angle are not identical.

It should be especially appreciated that separate illumination of areference marker and a substrate will have numerous advantages. Forexample, known optically analyzed micro arrays typically require thatthe focal plane or focal point for detection of a labeled analyte mustbe independently determined for each of the labeled analytes bound tothe micro array, which almost always necessitates illumination of thefluorescent marker of the analyte. Consequently, and especially wherethe adjustment to the optimal focal plane or point is relatively slow(typically up to about one minute per spot), photobleaching (andconcomitantly loss of actual signal) of the fluorophor is all butinevitable. Moreover, individual focal adjustments tend to increaseanalysis time dramatically, especially where the density of the labeledanalytes is relatively high.

In contrast, contemplated devices employ a light source that illuminatesone or more reference spots through the housing wherein the illuminationlight is preferably at least 20 nm different from the illumination lightof the substrates. Especially contemplated configurations provide adarkfield illumination of the reference spots. Thus, the intensity ofthe illumination light of the reference spots may be significantlyreduced, thereby reducing the likelihood of photobleaching of labeledanalytes bound to the substrates.

Moreover, where the reference marker(s) are in predetermined positionrelative to the substrates, illumination of the reference marker may beemployed to direct the multi-substrate chip into the focal plane of anoptical instrument that acquires the optical signal from labeledanalytes coupled to the substrates on the multi-substrate chip.Consequently, it is contemplated that refocusing for each of thesubsequent pixels in the multi-substrate chip may be partially, if notentirely avoided, thereby significantly reducing measurement time forthe entire multi-substrate chip. Determination of the correct focalplane may be provided by acquisition of more than one reference marker,and it is especially contemplated that each multi-substrate chipincludes at least four reference markers.

While it is generally preferred that the entire housing is completelylight-permeable, it should also be appreciated that only portions (e.g.,channels through the housing with or without lenses) may belight-permeable for illumination of the reference markers.Alternatively, and especially where the housing is light-impermeable, itis contemplated that the reference markers are illuminated withconventional darkfield illumination, or in an illumination in which thereference marker is illuminated by a first light source at a firstangle, and wherein at least one of the plurality of substrates isilluminated by a second light source at a second angle (preferably witha difference in angle of greater than 45 degrees).

Suitable reference markers include all known molecules or compositionsthat produce an optically detectable signal (i.e. light emission orabsorption) upon illumination. Therefore, all known chromophores andfluorophores are especially contemplated. Furthermore, it should beappreciated that the reference marker may also include achemiluminescent portion that emits an optically detectable signal underpredefined reaction conditions. Such reaction conditions may, forexample, be generated by addition of suitable reagents to the cavity ofthe device and are well known to a person of ordinary skill in the art.

Especially preferred light sources include various lasers, however, itis generally contemplated that all light sources known for photonexcitation (e.g., fluorescence, phosphorescence) are suitable for useherein. There are numerous suitable light sources known in the art, anda exemplary collection of such sources may be found in FluorescenceMethods and Protocols (Methods in Molecular Biology) by Dan Sackett(Humana Press; ISBN: 0896035441), or Fluorescence Microscopy andFluorescent Probes by Jan Slavik (Plenum Pub Corp; ISBN: 0306460211).However, it is particularly preferred that where lasers are employedthat the difference in wavelength between the first and second laser(for illumination of reference marker and substrate) is at least 20 nm.

Still further, suitable housings and/or multi-substrate chips mayinclude one or more registration markers, barcodes, and/or standardsthat may be read manually or automatically. For example, it iscontemplated that manually readable markers include imprinted orotherwise affixed serial numbers, type of substrates on the chip,supplier telephone numbers, etc. Automatically readable referencemarkers may include bar codes or one or more colored or fluorescent tagsthat may encode a particular piece of information.

With respect to the size of the housing, it is contemplated that aparticular size is not limiting to the inventive subject matter.However, preferred sizes are typically sizes in which the longestdimension of the housing is less than 10 inches, more preferably lessthan 5 inches, even more preferably less than 2 inches, and mostpreferably 1 inch and even less.

Consequently, it is contemplated that the volume of suitable cavitiesmay vary considerably. However, preferred cavities will have a volume ofless than 20 ml, more preferably between 0.01 ml and 1 ml, and mostpreferably between 0.01 ml and 1 ml. With respect to the shape ofsuitable cavities it is contemplated that any reasonable shape will beappropriate so long as such shape will accommodate the multi-substratechip at least in part. For example, suitable cavities may have a round,elliptical, or square shape. Similarly, the walls of the cavity may beperpendicular to the surface of the multi-substrate chip or in any angle(preferably between 45 degrees and 89 degrees). Therefore, depending onthe size and configuration of the cavity, the wall(s), and themulti-substrate chip, it is contemplated that the multi-substrate chipmay be located in various positions of the cavity. However, it isgenerally preferred that the multi-substrate chip is disposed at thebottom of housing (which may or may not be a base element).Alternatively, however, the multi-substrate chip may also be attached tothe housing such that the multi-substrate chip will be in a positionother than at the bottom of the cavity (e.g., suspended from the wallsof the cavity).

It is still further contemplated that the cavity may be in fluidcommunication with one or more liquid manipulation ports, wherein suchports may be configured to receive a pipette tip for addition and/orremoval of fluids. Alternatively, contemplated liquid manipulation portsmay also be channels or through-holes in the housing to add and/or draina fluid. While not especially preferred, contemplated liquidmanipulation ports may further include reservoirs that retain reagentsor other test related fluids, or provide structures to increase/decreasenon-linear flow of a liquid, or structures to accelerate or decelerateflow of a liquid, or structures to mix a fluid with another fluid.

In a further preferred aspect of contemplated devices, the cavity is influid communication with an overflow compartment, and it is especiallypreferred that the cavity is in fluid communication with the overflowcompartment only when the cavity contains liquid in a volume that isgreater than the predetermined volume of the cavity. Thus, contemplatedoverflow compartments may comprise a channel or a through-holepositioned such that fluid is only received from the cavity when thelevel of the fluid reaches a predetermined height. Additionally,suitable overflow compartments may include one or more overflow liquidmanipulation ports, and the same considerations as for the liquidmanipulation port(s) as described above apply for contemplated overflowliquid manipulation ports. In a particularly preferred aspect, theoverflow compartment is a channel that at least partially surrounds thecavity and includes at least one overflow liquid manipulation port.

In a further particularly preferred aspect of the inventive subjectmatter, the multi-substrate chip is disposed at or near the bottom ofthe cavity and the cavity is in fluid communication with an overflowcompartment only when the cavity contains liquid in a volume that isgreater than the predetermined volume of the cavity. Viewed from anotherperspective, it should be especially recognized that in such devices thevolume of a fluid in the cavity may be maintained at a constant valuewithout prior determination of the amount of fluid that is already inthe cavity. A constant volume of fluid is particularly desirable whereillumination of a sample, or detection of an optical signal from themulti-substrate chip is performed through a layer of a fluid, since theheight of the fluid layer is predetermined and substantially constant(i.e., changes typically less than +/−5%, more typically less than+/−2%) in such cavities.

FIG. 4 illustrates some of the advantages of contemplated cavities thatare in fluid communication with an overflow compartment. Here, amulti-substrate test device 400 has a cavity 410 in fluid communicationwith the overflow compartment 420 (when the fluid volume in the cavityexceeds the volume of the cavity). Laser beam 432 from laser. 430(typically from a confocal microscope; not shown) is deflected at cavityfluid surface 412. The angle of deflection is predominantly determinedby the refractive index of the fluid and the angle of laser beam 342relative to the surface 412. Regardless of the angle, however, it shouldbe appreciated that the horizontal deviation D1 and D2 will, among otherthings, be determined by the length of the path that the light willtravel through the fluid. Thus, a predetermined volume of the cavity(and therefore a predetermined height of the fluid) will significantlyreduce, if not entirely eradicate misillumination of positions on themulti-substrate chip. Moreover, providing a constant fluid volume overthe multi-substrate chip will circumvent most of the problems withattempts to remove fluid prior to detection of an optical signal (e.g.,incomplete draining, entrapped air bubbles, etc.).

In a further preferred aspect of contemplated devices, the cavity isformed at least in part by the housing and a base element, and it isespecially contemplated that the housing and the base element areremovably coupled to each other (e.g., via pins, screws, etc.). However,in alternative aspects, the housing may also be permanently coupled tothe base element. Thus, the multi-substrate chip in at least some of thepreferred devices may be coupled (directly or indirectly) to the baseelement. Direct coupling means that the carrier of the multi-substratechip is attached to the base element, whereas indirect coupling meansthat there is at least one additional layer between the multi-substratechip and the base element.

In yet further preferred aspects, the cavity is an open cavity, whereinthe term “open cavity” as used herein refers to a cavity in the housingthat is accessible from a point outside the housing without passingthrough a wall of the housing or without passing through a channel thatconnects the cavity with the outside of the housing.

With respect to the material of the base element, it is contemplatedthat any material suitable for (a) supporting the multi-substrate chipand (b) attaching the base element to the housing is considered suitablefor use herein. However, it is generally preferred that the base elementcomprises a material (and is configured) to transfer thermal and/orultrasound energy to the multi-substrate chip and/or a fluid in thecavity. Consequently, particularly preferred materials include metals(e.g., aluminum), ceramics, and synthetic polymers.

While not limiting to the inventive subject matter, it is generallypreferred that the base element has at least one, more preferably twoguide elements that will assist in automated handling of contemplatedanalytical devices. For example, contemplated guide elements includeindentations or protrusions from the base element, but may also includemagnetic spots or elements engaging with an actuator (e.g., hooks,loops, etc.). Thus, contemplated devices may comprise a housing with afirst width and a base element with a second width, wherein the firstwidth is smaller than the second width.

In yet further alternative configurations, suitable analytic devices mayinclude more than one multi-substrate chip (with at least one chip beingat least partially disposed in the cavity). For example, where cellextracts are analyzed in such devices, a first multi-substrate chip maybe employed to analyze a nucleic acid population while a secondmulti-substrate chip may be employed to analyze a polypeptide population(see FIG. 2B). Alternatively, and especially where hybridizationconditions vary between or among multiple multi-substrate chips,multiple cavities and with multiple multi-substrate chips may beemployed (e.g., with one chip per cavity), wherein at least one of thechips is at least partially disposed in the respective cavity (see FIG.2A).

Preferred matrices are multi-functional matrices that include inaddition to the crosslinker at least one further additive that isspecific to a particular configuration or test condition. Suitableadditives may impart selected characteristics and especiallycontemplated additives include a buffer (e.g., to adjust stringency to aparticular level, to modify pH to a particular value, etc.), a humectant(e.g., to maintain or adjust matrix hydration), a light blocking agent(e.g., to suppress carrier autofluorescence), or a surfactant (e.g., toimprove matrix adhesion to the carrier).

Furthermore, suitable matrices may include multiple layers, which arepreferably chemically distinct layers. For example, a first layer mayinclude a detergent to improve adhesion to the carrier, while a secondlayer may include a light blocking agent to reduce carrierautofluorescence, and a third layer includes the crosslinker that isemployed to couple the substrate to the carrier. It is generallycontemplated that any crosslinker is suitable that retains (covalentlyor non-covalently) a modified or unmodified substrate, it isparticularly preferred that the crosslinker comprises a molecule thatbinds biotin with a K_(D) of no greater than 10⁻⁵M. Thus, suitablecrosslinkers include avidin, streptavidin, and antibodies againstbiotin. Furthermore, it is contemplated that suitable crosslinkers inthe matrix may also be employed to bind a reference marker or referencesubstance against which position or amount of optically detected signalmay be calculated.

Moreover, it should be especially appreciated that in preferred aspectsthe matrix (e.g., agarose, gelatin, or polyacrylamide) is coated ontothe carrier by methods well known in the art. Therefore, problemsassociated with uneven carrier surfaces are generally avoided.Furthermore, by addition of additives, undesirable signal interferencefrom the carrier can be substantially reduced, if not eliminated.

In yet another aspect of the inventive subject matter, it should beappreciated that a plurality of contemplated analytical devices may beincluded into a magazine to facilitate reloading of an analyzer with anumber of multi-substrate chips. While the arrangement of the analyticaldevices in the magazine may vary considerably (e.g. linear as a band orchain of devices, two-dimensional as an array of devices, orthree-dimensional as a roll of devices), it is generally preferred thatcontemplated devices are stacked such that a base element of a firstdevice is disposed above a housing of a second device. A guide in amagazine may engage with a guide element in contemplated devices, and aweight or a spring on top of the devices may provide the mechanicalforce to sequentially advance the devices to the bottom of the magazine.An exemplary magazine is depicted in FIG. 3, in which the magazine 300includes a plurality of analytical devices 300A.

In operation, an analytical device according to the inventive subjectmatter is provided (e.g., in a magazine, or manually-inserted into ananalyzer), and in one step, the plurality substrates is contacted withat least one fluid selected from the group consisting of a sample fluid(e.g., whole blood, cell extract, etc.), a reagent fluid (e.g. high-saltfluid to adjust stringency), a wash fluid (e.g., water), a labeled probe(e.g., nucleic acid or antibody) and/or a detection fluid (e.g.,calorimetric or luminogenic substrate), preferably when themulti-substrate chip is in a substantially horizontal position (i.e., nomore than ±15 degrees from horizontal, more typically no more than ±8degrees from horizontal, and most typically no more than ±3 degrees fromhorizontal). In a further step, it is contemplated that at least one ofthe plurality of substrates binds an analyte from the fluid (e.g.,sample fluid), wherein binding of the analyte is detected in asubstantially horizontal position.

Therefore, in particularly contemplated aspects of the inventive subjectmatter, preferred devices will include a first light source thatilluminates one or more reference markers, wherein illumination of thereference markers is employed to determine the focal plane of theoptical device (typically a confocal microscope) that analyses themulti-substrate chip. Once the correct focal plane is determined,analysis of the probes, samples, or other molecules on themulti-substrate chip may then proceed without further focusing by usinga second light source (typically from the confocal microscope). Suchpredetermination of the focal plane is particularly advantageous whererelatively large numbers of individual measurements are employed. Thus,it should be recognized that analysis of a plurality of substrates on amulti-substrate chip may be performed significantly faster than withknown devices since re-focusing from one substrate to the next tooptimize signal strength may be omitted. Moreover, it should berecognized that determination of the focal plane using reference markerson the multi-substrate chip (preferably using an illumination wavelengthother than the wavelength for illumination of the substrates) willadvantageously reduce, if not eliminate photo-bleaching of fluorophores.While it is generally preferred that illumination of the referencemarker(s) is performed with a light-emitting diode, other light sources(incandescent or fluorescent) are also considered appropriate.Furthermore, where appropriate, first and second light source may beidentical.

Thus, specific embodiments and applications of improved substrate chipsdevices have been disclosed. It should be apparent, however, to thoseskilled in the art that many more modifications besides those alreadydescribed are possible without departing from the inventive conceptsherein. The inventive subject matter, therefore, is not to be restrictedexcept in the spirit of the appended claims. Moreover, in interpretingboth the specification and the claims, all terms should be interpretedin the broadest possible manner consistent with the context. Inparticular, the terms “comprises” and “comprising” should be interpretedas referring to elements, components, or steps in a non-exclusivemanner, indicating that the referenced elements, components, or stepsmay be present, or utilized, or combined with other elements,components, or steps that are not expressly referenced.

1. An analytical device comprising: a housing at least partiallyenclosing a multi-substrate chip, wherein the multi-substrate chipincludes a gel matrix that comprises a light-blocking material, whereinat least two positional markers and a plurality of substrates arecoupled to the gel matrix, and wherein the gel matrix comprises amaterial selected from the group consisting of agarose, gelatin, andpolyacrylamide; wherein the housing is configured to allow illuminationof the at least two positional markers from a first position above thegel matrix by a first light source at a first angle such that tworeference signals are produced; wherein the housing is furtherconfigured to allow illumination of a labeled analyte bound to at leastone of the plurality of substrates from a second position above thematrix by a second light source at a second angle such that an analytesignal is produced, wherein the first angle and the second angle are notidentical; wherein the positional markers are configured to producefirst fluorescence signals and wherein the labeled analyte is configuredto produce a second fluorescence signal; wherein the at least tworeference markers and each of the plurality of substrates are disposedon the multi-substrate chip in predetermined spatial relationships toeach other to allow determination of a correct focal plane for each ofthe plurality of substrates; wherein the housing is still furtherconfigured to allow acquisition of the reference signals and the analytesignal when the multi-substrate chip is in a substantially horizontalposition; wherein the multi-substrate chip is disposed within an opencavity that is at least partially formed by the housing, and wherein thecavity has a predetermined volume of between 0.01 ml and 10 ml; andwherein the open cavity is configured to allow reagent addition andillumination of at least one of the plurality of substrates from a pointoutside the housing without passing through a wall of the housing orwithout passing through a channel that connects the open cavity with anoutside of the housing.
 2. The analytical device of claim 1 wherein atleast a portion of the housing is translucent.
 3. The analytical deviceof claim 1 wherein the housing is configured to allow illumination ofthe at least two positional markers in a darkfield.
 4. The analyticaldevice of claim 1 further comprising a third positional marker.
 5. Theanalytical device of claim 1 wherein the housing is configured to allowillumination at a difference between the first angle and the secondangle of at least 45 degrees.
 6. The analytical device of claim 1further comprising at least one of a barcode, a standard, and aregistration marker.
 7. The analytical device of claim 1 furthercomprising an overflow compartment, wherein the open cavity and theoverflow compartment are configured such that the open cavity is influid communication with the overflow compartment when the open cavitycontains a liquid in a volume that is greater than the predeterminedvolume.
 8. The analytical device of claim 1 wherein the housing includesa base element that is configured to transfer at least one of thermalenergy and ultrasound energy to at least one of the multi-substrate chipand a fluid disposed in the cavity.
 9. The analytical device of claim 1wherein at least one of the plurality of substrates has a structure thatallows binding of an analyte from a sample fluid, and wherein thehousing is configured to allow detection of binding of the analyte whenthe multi-substrate chip is in the housing and in a substantiallyhorizontal position.
 10. An analytical device comprising: a housinghaving at least one open cavity, and a multi-substrate chip at leastpartially disposed in the open cavity; wherein the multi-substrate chipincludes a gel matrix that comprises a light-blocking material, whereina plurality of substrates and at least two positional markers arecoupled to the gel matrix, and wherein the gel matrix comprises amaterial selected from the group consisting of agarose, gelatin, andpolyacrylamide; wherein each of the substrates and positional markersare in predetermined positions relative to each other such as to allowdetermination of a correct focal plane for each of the substrates,wherein at least one of the plurality of substrates is non-covalentlycoupled to a carrier in a predetermined position via a crosslinker thatis disposed in the gel matrix; and wherein the open cavity is configuredto allow reagent addition and illumination of at least one of theplurality of substrates from a point outside the housing without passingthrough a wall of the housing or without passing through a channel thatconnects the open cavity with an outside of the housing.
 11. Theanalytical device of claim 10 wherein the matrix is formed from at leasttwo distinct agarose layers.
 12. The analytical device of claim 10wherein the matrix comprises at least one additive selected from thegroup consisting of a buffer, a humectant, and a surfactant.
 13. Theanalytical device of claim 10 wherein the crosslinker comprises amolecule that binds biotin with a K_(D) of no greater than 10⁻⁵M. 14.The analytical device of claim 10 further comprising a base elementcoupled to the housing, wherein the housing has a first width and thebase element has a second width, and wherein the first width is smallerthan the second width.
 15. The analytical device of claim 14 wherein thebase element has a lateral cutout in the base element that is configuredto operate as a guide element.
 16. The analytical device of claim 10wherein the housing is configured to allow contacting of the pluralityof substrates with at least one fluid selected from the group consistingof a sample fluid, a reagent fluid, a wash fluid, and a detection fluidwhen the multi-substrate chip is in a substantially horizontal position.17. The analytical device of claim 16 wherein at least one of theplurality of substrates has a structure that allows binding of ananalyte from a sample fluid, and wherein binding of the analyte isdetected when the multi-substrate chip is in a substantially horizontalposition.
 18. A magazine comprising a plurality of analytical devicesaccording to claim 1 or claim 10, wherein the plurality of analyticaldevices are stacked such that a base element of a first device islocated above a housing of a second device.